Effects of dietary protein level on small intestinal morphology, occludin protein, and bacterial diversity in weaned piglets

Abstract Due to the physiological characteristics of piglets, the morphological structure and function of the small intestinal mucosa change after weaning, which easily leads to diarrhea in piglets. The aim of this study was to investigate effects of crude protein (CP) levels on small intestinal morphology, occludin protein expression, and intestinal bacteria diversity in weaned piglets. Ninety‐six weaned piglets (25 days of age) were randomly divided into four groups and fed diets containing 18%, 20%, 22%, and 24% protein. At 6, 24, 48, 72, and 96 h, changes in mucosal morphological structure, occludin mRNA, and protein expression and in the localization of occludin in jejunal and ileal tissues were evaluated. At 6, 24, and 72 h, changes in bacterial diversity and number of the ileal and colonic contents were analyzed. Results showed that structures of the jejunum and the ileum of piglets in the 20% CP group were intact. The expression of occludin mRNA and protein in the small intestine of piglets in the 20% CP group were significantly higher than those in the other groups. As the CP level increased, the number of pathogens, such as Clostridium difficile and Escherichia coli, in the intestine increased, while the number of beneficial bacteria, such as Lactobacillus, Bifidobacterium, and Roseburia, decreased. It is concluded that maintaining the CP level at 20% is beneficial to maintaining the small intestinal mucosal barrier and its absorption function, reducing the occurrence of diarrhea, and facilitating the growth and development of piglets.


| INTRODUC TI ON
The development of the piglet breeding industry plays an important role in the development of China's agricultural economy. Due to the physiological characteristics of piglets, the morphological structure and function of the small intestinal mucosa change after weaning, which easily leads to diarrhea in piglets (Kwon et al., 2014;Wijtten et al., 2011). Therefore, antibacterial growth promoters are commonly used to improve the growth and development of weaned piglets and to inhibit the reproduction of pathogenic bacteria. However, with the continuous increase in drug resistance risk, it is urgently important to identify another method of piglet husbandry. Protein is an indispensable nutrient for the growth of piglets, but piglets are very sensitive to crude protein (CP) levels. Lower CP levels reduce villus height and crypt depth in the small intestine, affect the balance of microbiota, decrease the digestion and utilization of proteins in the intestine, and decrease the growth performance of piglets (Luo et al., 2015;Peng et al., 2016Peng et al., , 2017, whereas excessive CP levels result in a large amount of undigested and utilized proteins entering the intestine and fermenting spoilage and increase the pH in the stomach and small intestine, thereby providing suitable conditions for colonization of the intestine by pathogens and disrupting the balance of intestinal bacteria, leading to diarrhea (Gao et al., 2020;Zhang et al., 2020).
As one of the organs connected to the outside world, the small intestine is continuously exposed to protein antigens and to bacteria and their degradation products, and it prevents these harmful substances from entering other parts of the body through its barrier function. The small intestinal mucosal barrier includes a mechanical barrier, a biological barrier, an immune barrier, and a chemical barrier. The mechanical barrier is primarily composed of small intestinal mucosal epithelial cells and intercellular tight junctions and is important to the small intestinal mucosal barrier.
Tight junctions, the primary components of the intestinal cell barrier, are composed of the tight junction proteins occludin, claudin, and ZO-1; these proteins function to strengthen intercellular junctions, avoid cell damage, and resist invasion by harmful substances and pathogenic microorganisms (Zihni et al., 2016). Occludin proteins play an important role in maintaining the integrity of tight junctions, as well as in maintaining small intestinal permeability (Buckley & Turner, 2018;Shil et al., 2020;Teng et al., 2020). Lochhead et al. found that (Lochhead et al., 2010) the outer loop of occludin protein is directly inserted into tight junctions and that the outer loop and the transmembrane portion interact with tight junctions; in this way, the membrane permeability at the junction site is reduced, and free access of macromolecules is blocked, to achieve barrier protection.
Under pathological conditions, occludin protein produces a contraction phenomenon and moves into the cytoplasm, resulting in the expansion of intercellular pores and destruction of the integrity between cells and increasing the translocation of macromolecules, toxins, and bacteria, which can easily lead to diarrhea (Khounlotham et al., 2012). Biological barriers to intestinal bacteria, which are the first barriers through which animals defend themselves against foreign pathogens, not only resist invasion by pathogens, participate in the metabolic synthesis of nutrients, and provide nutrition for the body but also regulate the host's intestinal immune system, interact with each other, and jointly maintain the homeostasis of the small intestinal microbial environment (Turkez et al., 2012). Weaning of piglets causes changes in intestinal microflora, reduced bacterial diversity, loss of appetite, diarrhea, and other phenomena; therefore, nutrients should be reasonably supplemented to improve the animals' performance during this period.
At present, diarrheal disease has been the cause of high mortality in children (Liu et al., 2019;Taborda et al., 2018). How to control infant diarrhea by regulating the level of protein has been a focus of recent research (Gao et al., 2020). The piglet model has become the best model for human nutrition. The growth of piglets is related to a variety of factors, such as a daily three-meal pattern and CP levels (Xie et al., 2020). In previous experiments, we found that 20%-24% CP would cause diarrhea in weaned piglets in the short term . In order to find a more appropriate CP level for piglet development in the short term, we not only investigated how changes in CP levels affect the morphological structure and the expression and distribution of occludin protein in the intestine of weaned piglets, but also explored the relationship between intestinal bacterial and diarrhea. In addition, we hope that provides a reference for the level of protein intake in infants.

| Animals and experimental design
Ninety-six weaned Du × Long × Large ternary crossbred piglets (25 days of age, and initial weight of 5.99 ± 1.07 kg) were purchased from Zhiping Farm in Qionglai City, Sichuan Province and randomly divided into four groups with four replicates of six pigs each. After 7 days of adaptation to feeding, they were fed diets with CP levels of 18%, 20%, 22%, and 24%. Each piglet was individually housed, and the piglets in each group had free access to food and water.
The piglets were housed in fully enclosed enclosures with leaky TA B L E 1-1 Sample collection number floors, teat-type drinkers, and adjustable stainless-steel tanks. The room temperature was controlled at 28-30℃, the relative humidity was controlled at 60%-70%, and the pig house was regularly ventilated. The environment and the appliances within the pig house were cleaned and disinfected before the test. Disinfection, deworming, and immunization were performed regularly according to the procedures of the pig farm during the entire test period.

| Histopathological observation of the small intestine
Preparation of tissue sections: After the jejunum and ileum were fixed for 24 h, the tissues were trimmed and embedded, dehydrated

| Immunohistochemical observation of small intestinal tissue
For immunohistochemistry, the embedded wax blocks of jejunal and ileal tissues were sliced, baked, deparaffinized, and rehydrated.
After the sections were treated with 3% H 2 O 2 for 15 min, they were immersed in a beaker filled with 0.01 mol/L citric acid buffer and placed in a 96℃ water bath to slowly reach a temperature of 95℃.  (100 μmol/L). The mixed solution was shaken well and stored at −20℃.

| Detection of occludin mRNA in the small intestine
For use, the primers were diluted with sterile ultrapure water to the working concentration (10 μmol/L). The relative expression levels of occludin mRNA in the jejunum and ileum were measured by RT-qPCR.

| Detection of occludin protein expression in the small intestine
According to the instructions provided with the animal whole protein extraction kit, total protein was extracted from jejunum and Electrophoresis on 2% agarose gels was used to detect the PCR products; equal amounts of the samples were combined according to the concentration of PCR products and mixed well; electrophoresis was then used to detect the PCR products, and the target bands were recovered. A library was constructed using the TruSeq ® DNAPCR-Free Sample Preparation Kit library construction kit, and the constructed library was quantified by Qubit and Q-PCR. After assessment of the quality of the library, double-end sequencing was performed using HiSeq2500PE250.

| Determination of the copy number of Lactobacillus, Bifidobacterium, Clostridium difficile, E. coli, and Roseburia
The 16S rDNA sequences of various strains were searched on GenBank, primer design was performed using Primer Express 5.0 (Table 1- The identity of the bacterium was confirmed. Each plasmid was extracted with a small amount of plasmid extraction kit and used as the standard. The extracted plasmids were identified by common PCR according to their respective primers and reaction procedures to check the size and integrity of the bands. The concentration of the standard (plasmid) was measured on a Nanodrop 2000. The copy number of the positive plasmid was calculated according to the following formula: plasmid copy number (copies/μl) = DNA concentration (ng/μl) × 6.02 × 10 23 (copies/ mol) × 10 −9 /[plasmid length (dp) × 660 (g/mol·dp)], and the known pMD19-T vector was 2692 bp in length.
Absolute RT-qPCR quantification was performed on a CFX96 Real-Time PCR System using the various primers, and dissolution curves were automatically generated after the reaction ended.

| Statistical analysis
The data for each sample were split from the offline data according to the Barcode sequence and PCR amplification primer sequence, and the reads of each sample were spliced using FLASH after truncating the Barcode and primer sequences. The resulting spliced sequences were the original Tags data (Raw Tags); the spliced Raw Tags required strict filtering (Bokulich et al., 2013) to obtain highquality Tags data (Clean Tags). The Tags obtained through the Tags quality control process of Qiime (Caporaso et al., 2010) need to be processed to remove chimeric sequences; these are detected by alignment with the species annotation database, and, finally, the chimeric sequences are removed to obtain the final effective data (Effective Tags).
Using Uparse software, the Effective Tags of all samples were clustered for Operational Taxonomic Units (OTUs) with 97% identity; the most abundant sequence in each OTU was then selected as F I G U R E 1-1 0 The pathological injury of ileum in each group on 96 h after feeding. (×200). (a-d represents the groups of different CP level 18%, 20%, 22%, 24%, respectively. The arrow points to the villus injury site.) the representative sequence of that OTU. Species annotation was performed on OTUs' representative sequences, and species annotation analysis was performed with the Mothur (Edgar, 2013) method with the SSU rRNA database of SILVA at a threshold of 0.8-1 to obtain the taxonomic information corresponding to each OTU. QIIME software was used to construct a dilution curve for the number of sequences and the corresponding number of OTUs. The dilution curve was prepared by randomly selecting a certain amount of sequencing data from the sample, counting the number of species the data represented (i.e., the number of OTUs), and constructing a curve based on the amount of sequencing data drawn and the corresponding number of species. The alpha diversity index (Chao1, Shannon, Simpson, ACE) was calculated using IIQME software (Version 1.9.1).
PCA analysis of community composition structure at the genus level was performed using R software.
The data were collated using Excel, and the results were analyzed by one-way analysis of variance using SPSS Statistics 22 statistical software with p < .05 as the criterion for discriminating significant differences. The results are expressed as the mean ± standard deviation.

| Histopathological changes in the jejunum
In Figures 1-1  In summary, CP at the 20% level caused less morphological damage to the jejunum and ileum of piglets. The edge of the villous epithelium was smooth, the length was uniform, and the epithelial cells were neatly arranged. In addition, crypts were also clearly visible.

| Effect of CP level on the ultrastructure of the ileal epithelium in weaned piglets
It can be seen in Figure 2-1 that at 6 and 24 h after feeding, the ileal microvilli of the animals in the 20% CP group were densely and very neatly arranged and were perpendicular to the top of the cells; the cells contained abundant organelles and showed no abnormalities

Note:
The same letter of shoulder in peer data indicates no significant difference (p > .05), the same letter but different case indicates significant difference (p < .05), different letters indicate extremely significant difference (p < .01), and the same data footmark is consistent with the shoulder marking method.

| Immunohistochemical results in the jejunum
From

| Effect of CP level on relative occludin mRNA expression in weaned piglets
3.4.1 | Effect on occludin mRNA expression in the jejunum As shown in Table 2-3, the relative expression of occludin mRNA in the jejunum at 6 and 24 h after feeding was highest in the 20% and 22% CP groups; the expression in those groups was very significantly higher than that in the 18% and 24% groups (p < .01), in which the relative expression of occludin mRNA in the jejunum was low.
At 48, 72, and 76 h after feeding, the relative expression of occludin mRNA was highest in the 20% CP group; at these times, the relative expression of occludin in this group was very significantly higher or significantly higher than that in the 22% CP group (p < .01 or p < .05).

| Effect on occludin mRNA expression in the ileum
From Table 2-4, it can be seen that the relative expression of occludin mRNA in the ileum at 6, 24, and 72 h after feeding was highest in the 20% CP group; at those times, it was very significantly higher than that of the 22% CP group (p < .01). After 48 and 96 h of feeding, the relative expression of occludin mRNA was highest in the 18% CP group; in that group, it was very significantly higher than in the 22% CP group (p < .01). Among all of the experimental groups, occludin mRNA expression was lowest in the 22% CP group.
Above all, the expression of occludin mRNA in the jejunum and ileum is highest at 20% CP group.

| Effect of CP level on occludin protein expression in weaned piglets
3.5.1 | Effect on expression of the tight junction protein occludin in the jejunum As shown in Table 2-5 and Figure 4-1, the expression level of the tight junction protein occludin in the jejunum at 6-48 h after feeding was highest in the 20% CP group; in that group, it was very significantly or significantly higher than the levels in the other three groups (p < .01 or p < .05). At 72 h after feeding, the expression of occludin was highest in the 22% CP group; in that group, the level was very significantly higher than the levels in the other three groups

Note:
The same letter of shoulder in peer data indicates no significant difference (p > .05), the same letter but different case indicates significant difference (p < .05), different letters indicate extremely significant difference (p < .01), and the same data footmark is consistent with the shoulder marking method.
(p < .01). However, at 24-72 h after feeding, the expression of occludin was lowest in the 18% CP group; in that group, it was very significantly lower than the levels in the other three groups (p < .01).
3.5.2 | Expression of the tight junction protein occludin in the ileum As shown in Table 2-6 and Figure 4-2, the expression level of the ileal tight junction protein occludin at 6, 24, and 72 h after feeding was highest in the 20% CP group; in that group, the level was very significantly higher than the levels in the other three groups (p < .01).
Among the 18%, 22%, and 24% CP groups, the expression level in the 24% CP group was the lowest (except at 24 h). After 48 and 96 h of feeding, the expression of occludin was highest in the 18% CP group; in that group, it was very significantly higher than in the 20% and 22% CP groups (p < .01).
In conclusion, in the jejunum and ileum, the expression of occludin protein was higher in 18% CP after 96 h of feeding and in 20% CP than in the other groups.

F I G U R E 3 -6
The immunohistochemistry of ileum in each group on 6 h after feeding. (×400 As shown in Figure 5-1, the dilution curve of each sample eventually tends to be flat. From Figure 5-2, it can be seen that the boxplot position tends to be flat and that the confidence interval is decreasing.
In summary, rarefaction curves of each sample eventually tend to be flat, indicating that the sequencing data of this test are reasonable and can include most microorganisms in the sample. Among them, 22% CP had the highest gut species richness.

| Alpha and beta diversity analysis
Alpha diversity reflects microbial community richness and diversity within a sample. As shown in Tables 3-3 and 3-4, there were no significant differences in alpha diversity among the groups (p > .05), but the high protein level groups (22% CP and 24% CP) displayed numerically higher alpha diversity than the 20% CP group, and this was F I G U R E 3 -1 0

TA B L E 3 -2 Microbial sequencing of colonic chyme
relative content of these phyla accounted for 83.22%-97.94% of the total sequences); in sample B2, the relative content of Aspergillus was the highest. As shown in Table 4-1, after 72 h of feeding, the relative content of Firmicutes in the 22% CP and 24% CP groups was very significantly lower than that in the 18% CP and 20% CP groups (p < .01). After feeding for 24-72 h, the relative content of Proteobacteria gradually increased as the dietary protein level increased. The relative content of Bacteroidetes in the 24% CP group was significantly higher than that in the other three protein level groups after 24-72 h of feeding (p < .01). Figure 6-2 shows the 20 genera with high relative abundance in the test samples. As shown in Table 4-2, Lactobacillus (13.84%−64.84%) was the dominant genus in all samples, and its relative content showed a decreasing trend as the dietary protein level increased. After feeding for 72 h, the relative content of Lactobacillus in the 24% CP group was significantly lower than that in the 18% CP group (p < .05). The relative content of unidentified Clostridiales is second only to Lactobacillus spp. Except for the 18% CP group, the relative content of Actinobacillus showed a decreasing trend with increasing feeding time, and the relative content of Actinobacillus in the 20% CP group was significantly lower than that in the 22% CP and 24% CP groups (p < .05); however, its relative content in the 18% CP group was very significantly higher than that in the other three groups at 72 h of feeding (p < .01).

| Species composition of colonic bacteria
The species composition of colonic digesta at the phylum level is shown in Figure 6-3; 20 bacterial phyla and 1 archaeal phylum were obtained from each sample in this experiment. Firmicutes, Proteobacteria, and Bacteroidetes were the dominant phyla in all samples except sample H1 (the relative content of these phyla accounted for 88.44%-97.26% of the total sequences). As shown in Table 4-3, the content of Firmicutes first increased and then decreased as the feeding time increased. However, the relative contents of both Proteobacteria and Bacteroidetes first decreased and then increased with feeding time. At 24 h of feeding, the relative

TA B L E 3 -4
Microbial alpha diversity index of colonic chyme content of Bacteroidetes was significantly lower in the 22% CP group than in the 20% CP and 24% CP groups (p < .05). After 6 h of feeding, the relative content of Proteobacteria increased as the protein level in the diet increased, and the relative content of Proteobacteria in the 22% CP and 24% CP groups was very significantly lower than that in the 18% CP and 20% CP groups (p < .01). The relative content of Actinobacteria gradually decreased with increasing feeding time; at 6 h of feeding, the relative Actinobacteria content of the 24% CP group was very significantly higher than that of the other three groups, but after 72 h of feeding, it was very significantly lower than that of the 22% CP group (p < .01) and significantly lower than that of the 20% CP group (p < .05).  Note: Peer data shoulder letter with the same case means significant (p < .05), letter with different case means extremely significant (p < .01).
relative Lactobacillus content of the 20% CP group was significantly higher than that of the 22% CP group (p < .05). The relative content of unidentified Ruminococcaceae was second only to Lactobacillus spp., and the relative content of unidentified Ruminococcaceae in the 22% CP group was very significantly higher than that of the other three groups at 24 h of feeding (p < .01); however, after 72 h of feeding, the relative content of unidentified Ruminococcaceae in the 22% CP and 24% CP groups was very significantly lower than that of the 18% CP

| Copy number results for Lactobacillus, Bifidobacterium, Clostridium difficile, E. coli, and Roseburia
In Tables 5-1 and 5-2, after 6 h of feeding, the number of E. coli in the ileum was significantly lower in the 18% CP group than in the 22% CP and 24% CP groups (p < .05). After 24 h of feeding, the number of lactobacilli in the colon was significantly lower in the 22% CP and 24% CP groups than in the 20% CP group (p < .05), and the numbers of Bifidobacterium and Roseburia in the ileum of the 24% CP group were very significantly higher than those in the 18% CP and 20% CP groups (p < .01). After 72 h of feeding, the number of C. difficile in the colon of the 22% CP and 24% CP groups was very significantly higher than that in the 18% CP and 20% CP groups (p < .01). Above all, the animals fed the high-protein diets showed an increased change in the numbers of bacteria in the ileal contents and that the number of lactobacilli, bifidobacteria, and Roseburia in the small intestine showed a decreasing trend with increased feeding time, whereas the number of C. difficile and E. coli showed an increasing trend.

| DISCUSS ION
For the better development of animal husbandry production in China, it is very important to find CP levels that can solve the  (Eid et al., 2003). Instead, the morphology of the small intestinal mucosa will also change when the level of protein in the diet is altered. The results of the current study showed that feeding piglets with 20% CP diet reduced intestinal damage, such as clearly crypt and increased intestinal villi, which in turn was conducive to the absorption and utilization of nutrients by piglets. Tight junctions exist between adjacent cells and anastomose to form a continuous fishnet structure that acts to maintain intercellular polarity and prevent penetration, and occludin protein plays an important role in this process (Roxas et al., 2010;Sappington et al., 2003). In this work, it was found that the tight junction structure was blurred, the organelles were damaged, and the intercellular space was enlarged in the 22%-24% CP groups, while the ultrastructure of the ileal tissue was intact in the 20% protein group, except at 24 h. Experiments performed over the same period also revealed that the diarrhea score was significantly higher in the high-protein groups than in the 20% protein group (p < .05), consistent with the experimental results of Htoo (Htoo et al., 2007) and Opapeju (Opapeju et al., 2009 (Wu et al., 2015) and Gophna (Gophna et al., 2017). The reason for this may be that high levels of CP increase the expression of proinflammatory factors; upregulation of proinflammatory factors leads to decreased expression of occludin in the jejunum and ileum of piglets (Al-Sadi et al., 2009;Gao et al., 2020).
16S rDNA amplicon sequencing technology is widely used in the comparative analysis of differences in microbial community structure in the natural environment and in human and animal tissues (Matsuki et al., 2004;Yang et al., 2015). It can be seen from  (Roca et al., 2014) to affect the diversity of gut microbes in animals. In the current study, it was found that the Chao index of the ileum and colon of piglets increased with the increase in CP level at 24-72 h after feeding and that the alpha diversity index of the colon was significantly greater than that of the ileum, indicating that with the increase of CP level, the intestinal richness also increased, and it could be seen that the colon was the main site of microbial fermentation in piglets, consistent with the experimental results of Konstantinov (Konstantinov et al., 2004). High-throughput sequencing technology was used to study the changes in intestinal microbial bacteria in piglets, and the results were consistent with the results of previous studies (Zhou et al., 2020): Firmicutes, Bacteroidetes, and Proteobacteria were the dominant bacteria in the ileum and colon of piglets. Studies (Jonkers et al., 2012;Willing et al., 2011) have found that these three phyla are involved in nitrogen metabolism, that they secrete proteases and affect protein fermentation in the intestine, and that decreased content of these phyla can increase the likelihood of bacterial pathogen colonization.
Intestinal bacteria play a major role in protecting against pathogens, and an imbalance of bacteria can cause local pathology (Minty et al., 2019). Lactobacilli and bifidobacteria are beneficial bacteria in the intestine and can inhibit invasion of the intestine by pathogenic bacteria by fermenting food residues to produce acidic substances such as lactic acid to reduce intestinal pH or by adhering to intestinal epithelial cells (Kailasapathy & Chin, 2000). In this experiment, the relative contents of Lactobacillus and Bifidobacterium in the gut of weaned piglets showed a decreasing trend with increasing dietary protein levels, similar to the study of Ma (Peiling, 2019). The reason for this is that high levels of dietary protein disrupt the intestinal mucosa of piglets and affect its colonization by Lactobacillus and Bifidobacterium. Roseburia is a key bacterium in the gut that degrades dietary fiber (Kasahara et al., 2018). The results of this study showed that high dietary protein levels increased the number of Roseburia in the ileum and colon and are consistent with the results reported by Hooda (Hooda et al., 2013). This may be because in this experiment the CP level in the diet was adjusted primarily by changing the corn is often also accompanied by the growth of bacterial pathogens such as E. coli and C. difficile, and once the balance among the intestinal bacteria is broken, the proliferation of pathogens that produce toxins can lead to the occurrence of secretory diarrhea. However, there are very few reports on the effect of dietary protein levels on the number of intestinal C. difficile. We found that with the extension of feeding time, the number of C. difficile was very significantly higher in the 22% CP and 24% CP groups than in the 20% CP group (p < .01), and related studies (Tan et al., 2019) showed that the abundance of Fusobacterium increased in the piglet diarrhea model; therefore, we surmised that an abundance of C. difficile in the colon is an important factor causing diarrhea in weaned piglets fed highprotein diets. Studies (Jeaurond et al., 2008;Wellock et al., 2008) found that diets containing high levels of protein contribute to the reproduction of E. coli in the intestine and that they also stimulate E. coli to produce large amounts of fermentation byproducts that reduce intestinal barrier function integrity, consistent with the results of this experiment. Some studies have found that high-protein diets can increase the incidence of diarrhea in piglets infected with enterotoxigenic E. coli. Thus, the increase in dietary protein levels causes the number of pathogens in the intestine to increase, and the resulting decrease in the number of beneficial bacteria aggravates diarrhea in piglets.

| CON CLUS ION
Weaned piglets 25 days of age were fed corn-soybean meal-based diets with CP levels of 18%, 20%, 22%, and 24% for one week.